Orthotopic liver transplantation (OLT) is an effective therapeutic modality for the treatment of end-stage liver disease. However, ischemia/reperfusion injury (IRI), an exogenous Ag-independent component of the harvesting insult, remains one of the key limitations after OLT. Moreover, the quality of donor organs decreases along with the aging of general population and associated pathological conditions. This proposal is built upon the insights gained from our previous studies on a novel approach of combating organ IRI by locally inducing heme oxygenase-1 (HO-1) that protects against the severity of oxidative stress. Indeed, local HO-1 expression and functional response should be considered as a denominator of donor organ quality. We have also shown the role of innate immunity in liver IRI. Indeed, by utilizing a non-transplant mouse warm liver IRI model, we have documented that: (i) TLR4 activation mediates liver inflammation via IRF-3 pathway; (ii) CXCL10 regulates liver innate immune responses; (iii) Type-I IFN mediates synergy between Kupffer cells and hepatocytes, and (iv) endogenous TLR ligands are crucial in TLR4 activation-induced sensoring and IRI. To mimic the clinical scenario, we have recently developed a mouse model of prolonged liver cold preservation, followed by syngeneic OLT. We hypothesize that cross talk between the opposing pathways, i.e., HO-1 in the donor organ, and TLR4 in OLT recipients is instrumental in the mechanism of liver IRI. Our corollary hypothesis states that Nrf2, a bZIP transcriptional factor that regulates stress response/regulates cell redox balance, controls dysregulated HO-1 - TLR4 signaling during IRI. Aim 1. To analyze mechanisms by which suboptimal HO-1 deficient liver grafts affect IRI sequel and TLR4 signaling in OLT recipients. Livers from WT, HO-1 deficient (+/-; KO) and HO-1 overexpressing (Tg) donors will be stored for 18 h at 4 C, and then transplanted to syngeneic WT mice. We will study (i) whether HO-1 expression and by which donor liver cell type affect IRI and OLT outcome, and (ii) if and how the local HO-1 expression in OLT affects host TLR4 signaling. Aim 2. To dissect mechanisms by which modulation of recipient TLR4 signaling ameliorate IRI and improve the outcome of suboptimal OLTs. Livers from WT or HO-1 deficient (+/-; KO) donors will be stored for 18 h at 4 C, and then transplanted to syngeneic TRIF- or Type I IFN receptor (IFNAR)-KO mice. We will study (i) whether selective ablation of downstream TLR4 signaling in the host affect IRI sequel/OLT inflammation, and (ii) if HO-1- TLR4 cross talk can influence HMGB1-mediated IR inflammation and organ damage in OLT recipients. Aim 3. To analyze mechanisms by which Nrf2 signaling influence IRI in OLT recipients. Livers from donor mice that are Nrf2-deficient or Nrf2-overexpressing (hepatocyte-specific conditional disruption of the Keap1 gene, which represses Nrf2) will be stored for 18 h at 4 C, and transplanted to WT mice. By modulating oxidant (HO-1 siRNA) and inflammatory (anti-HMGB1/rHMGB1) responses, we will study the regulatory function of Nrf2 upon (i) HO-1 vs (ii) TLR4 signaling pathways during IRI in OLT recipients.